Antiviral agent fwh-775 and method of production

ABSTRACT

THE PRESENT INVENTION RELATES TO A NEW ANTIVIRAL SUBSTANCE. MORE PARTICULARLY, THE PRESENT INVENTION RELATES TO A NEW ANTIVIRAL SUBSTANCE OBTAINED FROM A FERMENTATION PROCESS UTILIZING A NOVEL MICROORGANISM OF THE SPECIES ASPERGILLUS NIGER. THE ANTIVIRAL SUBSTANCE HAS BEEN FOUND TO BE EFFECTIVE AGAINST BOTH DNA AND RNA VIRUSES IS LABORATORY ANIMALS.

United "States Patent Ofice 3,819,832 Patented June 25, 1974 s Claims ABSTRACT or :THECDISCLOSURE The'present invention relates to a new antiviral substance. More particularly, the present invention relates to a new antiviral substance obtained from a fermentation proc-'' ess utilizing a novel microorganism of the species Aspergillus niger. The antiviral substance has been found to be effective against both DNA and RNA virusesin laboratory animals. i

This application is a continuation-in-part of Application Ser. No. 64,025, tiiled July 20, 1970, now abandoned, which, in turn was a continuation application of Applical tion Ser. No. 616,159 filed Feb. 15, 1967, now abandoned, which, in turn was a continuation application of Application Ser. No. 547,167 filed May 3, 1966, now abandoned.

The present invention relates to a new antiviral substance which is obtained by'cultivating a new fungus which veniently used as a dilute solution for administration of the active substance which eliminates theproblem of cum- Fermentation broths containing FWH-77 5 are-produced by inoculating spores of ATTC No. 16508 into 'a suitable medium and then cultivating under aerobic' conditions. It has been established that 50 percent less activity bersome separation and purification steps. H

results in the FTC broth when fermented under anaerobic conditions rather than aerobic; FTC-activity in-the broth is not-increased by increasing the glucoseconcentration in the medium beyond 1-.2 milligrams/mgml. As a concentration of glucose is increased, the vfinal pH of the medium decreases and the activity in the broth is accordingly reduced. Agitated culture after 48 hours incubation has identical activity as that incubated for 6 days without agitation, Eleven media containing various amino acids were fermented (Table 2) and the results in bivo activity indicate that glutamic acid and phenylalanineareessentia1 V olate.

morphological studies have. determined is of the Aspergillus niger species and has the following characteristics: distinct thick walled and single celled foot cell: septate mycelium: globose, smooth, thick walled vesicle: primary and secondary strigmata anda round conidia. The organismwas isolated from swine embryo kidney tissue cultures infected with Batts'V 13, a swine entero virus.'A culture of the living organism has been deposited in the American Type Culture Collection, 12301 .Parklawn Drive, Rockville, Md., U.S.A., 20852 and assigned ATTC. No..16508 pursuant to the terms of contract No. 474 between Frank W. Horner, Limited assignee of the. instant application and the American Type Culture Collection. V

The active antiviral substance of the present invention believed to be an amino acid derivative of low molecular weight, hereinafter designated as FWI -I-'-775, is obtained by cultivating ATTC No.- 16508 :under aerobic conditions in an aqueous culture medium preferably at temperatures ranging from about 20 to'about The antiviral substance is found in the fermentation broth in minor amounts. Inasmuch as the substance of the present invention is extremely active againstviruses at very low concentrationsfthe fermentation ,brothitself can be confor antiviral activity.

The nature of the culture medium does not appear to be critcial. A suitable culture medium consists of Hanks balanced salt solution enriched with 0.5 percent lactalbumin hydrolysate (enzymatic) and 0.1 percentyeast- The temperature of the cultivation may be variedover a wide range of from about 20 C. to about 40 C. At temperatures below about 20 C. the growth rate of the microorganism is extremely slow whereas at temperatures above 40 C. the biosynthetic activity of the microorganism is reduced. A cultivation temperature of about 37 C. is preferred.

The time of cultivation is dependent on various factors such as temperature, method of incubation and the like, and can be varied over wide limits. When the cultivation is carried out by stationary incubation at the preferred temperature of 37 C., a cultviation time of from 6 to 15 days gives optimum results. On the other hand, if the cultivation takes place at the same temperature under agitated conditions, suitable results can be obtained in substantially shorter periods of from 2 to 4 days. The fermentation is carried out at a substantially neutral pH, for example, pH 6.0-8.0 and preferably at a pH of 6.5-7.0. It has been found that in general, depend-- ing on the characteristics on the culture medium used, the

"pH value changes substantially as the fermentation proceeds. For example, when the fermentation is carried out on the enriched Hanks balanced salt solution described above, the initial pH is about 6.4-6.5. When said fermentation is allowed to go to completion of pH is generally about 8.3-8.5.

The growth characteristics of. the microorganism are set forth in Tables l-3 which follows. Additionally, the changes in pH in the various fermentationbroths are set forth in Table 4 which follows.

V TABLE I Growth characteristics of Aspergillus nigervar, Joshi 16508 at 37 C. on solid media 24 hours 48hours 72l1ours 1 96 hours 168 hours Mediumsabourauddextrose; v I 7' V W v I W 7 p I m i Growth Slight-mo r Good- Good. Good- Good.

Color of irultin -.Greenish- (black) Black (greemsh)...,.. Black (greenish) Black (greenish).

Color on revers lightly yeliowish Sli(ghtly yellgiwipg Yieililotwishlgreyish Greyish/yellow.

- Y a nearmar ns.

Arrangement of fr i in 6 Concentric fr11l ting Concentric fruiting. Concentric trulting. Medium-r0 ekdoxa arr 2 Growt p g Sli ht Moderate Moderate-good Good Good-abundant.

Fruitin I 0-. h g ode ratmu nd? filsmuedant.

C l tfruiti 5.-.... Black ee C813; n revei si Colorl Slightly yelgllowishl, Yellow reverse Yellow reverse;

nearmar n. I -1 Arrangement of fruitin v TABLE IContinued 24 hours 48 hours 72 hours 96 hours I 168 hours Medium-Nutrient agar:

Growth Slight I Slight I Slight-moderate Moderate Moderate. w Fruiting.-. Slight fruiting.. Moderate fruiting- Do. Color of fruiting- Greenish black... Black (greenish) Black. Color on reverse Colorless.... Light greyish yellow.. ,Greyish.(yel1ow tint). I Greylsh yellow. Arrangement of fruiting I Medium-Potato dextrose agar: I I

Growth. Slight..- Moderate.. Moderate-good.. I Fruitin Slinrhf M0derate Color-... Black (greenish). Blank la Black.

Color on reverse Greyish white Grey... Grey (yellowish) Yellow'ishgrey Arrangement of fruitin Cnn mnfrir' Cnnnnntrir' Concentric. Medium-Malt extract agar: I I Growth Moderate-good Abundant Abundant Abundant Abundant.

Fruiting M0derate.. ..do-. I ...do- Do. Color of fruiting- Black (greenish) Black.... Black Black Black. I I Color on reverse.-. Yellowish Distinct yellow .-Strongly yellow--. Strongly yellow Deep yellow. 1 Arrangement of fruiting" Medium-Sabouraud maltose I A agar: .1"'. I I I I I .I.I Growth Slight-moderate-..-.... Moderate-....- Moderate Moderate Moderate. Fruiting g t- I .do..-..- Good-abundant- Abundant. Color of fruiting Greenish black Black (greenish). Black. I Color on reverse- I Ye1lowis h Yellowish Yellow i (rllligrgins I gree s Arrangement of fr i in I TABLE II Growth characteristics of Aspergillus niger var. Joshi 16608 at room temperature (21 C.) on solid media 24 hours 48 hours 72 hours 96 hours I I 168 hours Medium-Sabournuddex- I trose agar: I

Grow h Slight-mbderate.. Fruiting Slightdog. Color of fr iflm' Greenish (black) Black I Color on reverse- Yellow/yellow tint Yellow/yellow tint I around fruiting in around fruiting in i front. I I front. Arrangement of fruitin Cnnv nffir' Concentric. Medium-Czapekdox agar:

Growth Sli ht I Slight-moderate.. Moderate. Frulting-- Poor-slight..... Slight-moderate. Color of fruitin Black-(groenish)...... Black (greenish).- Color on rev I Distinct yellow] I I Distinct yellow/ yellow around yellow around I 1 fruitlngin front. 1. fruiting in front. Arrangement ofirniflnv I I I Medium-Nutrient agar: i A

Grow h -S1ight-rnn mf Moderate. Fruiting. Poor-slight. 0. II Color of fr Black. Color on reverse.-.- I Slight yellowish; Arrangement of fruiting I I Medium-Potato dextrose i agar: I r 7 Grow Y Slight-"moderate Slight-moderate Moderate. I I Fruiting I Slight--- -."Moderate -Do. Color of fr Greenish 5 Black Black. II Color on reverse. Yellowish with, Greyish'yell Yellow '(greyish).

.-- yellow tint around with yellow tint iruiting in front. I around fruiting in front. Arrangement of fruiting-... Medium-Malt extract agar: I I I Growth Slight-moderate Good Fruiting.. Morim- Good-abundant d Color of fruiting ,Black (greenish) Black. Black I, I Black. Color on reverse... I Yellow Distinct yellow/ Distinct'y'ellow/ Distinct deep yellow. yellow tintaroundv 1 ,yellowftint around fruiting area in v fruiting area in v v front. front. I Arrangement of fr iiimr I Medium-Sabouraud Malt- I v I I I I toseagar: 4 Grow h r Sli ht .Moderate.....;...;...'. Moderate. Fmiiinn I I I Slight-moderate DO. Color of fr Black( reen).'. Black. Color on reverse--.. Yellow yellow tint vYellow/yellow tin i v I I around the fruiting around thefrulting area infront. area in front. Arrangement oifrnilin w T E I Growth eharacteristicsof Aspergillus niger var. Joahi 16508 at,37 C. inliquid media 24 hours 5v 96 hours J a 192 hours"- Medium-Czapekdox broth: Growth as a whole.- Moderate growth. Moderate growth. Abundant growth.

Surface growth Surface/moderate Surface/moderate. Surface/moderate. Submerged growt Submerged/mil Submerged/mil. Submerged/um. Fru ting.-. Fruiting slight. Fruiting moder Fruiting abundant. Fruiting c010 Fruiting black. Fruiting bla c k. Changes in the me um colo N 0 change in In No change in me No change in medium color. Medium-Nutrient broth u for; 2-1:: P

Growth as a whole Poor growth- Poor-moderate growthl Moderate growth. I Surface growth.. Surface growt Surface'growth... Surface growth. I Submerged/mill.-. Submerged/nil]. 1" Fruiting slight-moderate.-.-. Frnitingmoderata. Fruiting black:.--...-. Fruiting black? No change in medium-color....::.. Medium darker TABIJE III- Contln ued e 24 hours 96 hours 192 hours Medium-Heart infusion broth:

Growth as a whole...- Abundant growth Abundant growth Abundant growth.

Surface growth... Surface growth. Surface growt Surface growth.

Submerged growt Submerged/hill Submerged/lull Submerged/hill.

Fruitin Fruiting poor. Fruiting mo slight Fruiting abundant.

Fruiting color Dark brown fruiti Black fruiting Black fruiting.

Changes on the medium color No change in medium color No change in medium colo Medium darker. Medium-Submerged maltose broth: 7

Growth as a whole Moderate growth Good growth Good growth.

Surface growth Surface growth Surface growth Surface growth.

Submerged growth Submerged/slight Submerged/slight.

Fruiting Fruiting moderate Fruiting abundant Fruiting abundant.

Fruiting color Black Black. Black.

Changes in the um color No change in medium color No change in medium color No change in medium color. Medium-Sabouraud dextrose broth:

Growth as a whole Moderate growth Moderate growth Good growth.

7 Surface growth Surface growth.-. Surface growth Surface growth.

Submerged growth- Submerged/slight Submerged/moderate... Submerged/moderate.- Fruiting Fruiting near periphery Fruiting moderate. Fruiting good. Fruiting color Bla k. Black Black.

Changes in the medium color No change in medium color/only No change in medium color/slight Medium light/yellowish tint.-

a yellow surface vring.

yellow surface ring.

TABLE IV.pH CHANGES Growth of Aspergz'llus nicer var. Joshi 16508 at 37 C. in broth After many attempts to isolate the antiviral active components from the crude broth were made by means of solvent extraction of the crude broth or the freeze-dried broth with a series of solvents including ethylacetate, benzene, ether and chloroform at acidic neutral and basic pH, it was determined that in all cases, activity remained in the water layers. It appeared that the active components are polar water soluble substances, probably low molecular weight amino acid derivatives.

It was observed that at pH 7.6 activity was not de- 40 stroyed upon heating at 60 C. for 3 hours, whereas all activity was lost at pH 1 with such a treatment. In order to obtain samples for testing the antiviral activity of FWH-775 in laboratory animals, the broths were pooled and freeze-dried to yield an off-white solid which consists of major amounts of inorganic salts of the culture medium and minor amounts of FWH-775.

The present antiviral substance is substantially stable at a pH in the range of 1-10 jand'is substantially heat stable. For example, the antiviral activity of broth samples containing FWH-775 and having a pH in the range of 7.0 8.5 remains substantially unchanged after heating said samples to 100 C. for a period of 10 minutes. 7 a v A typical method of carrying out'the process of the present invention by stationary incubation is described below:

Twelve one-liter flasks were each charged with about 400 ml. of enriched Hanks salt solution, inoculated with an aqueous spore suspension of the organism and stoppered with loose cotton plugs. p I 1 0 The composition of the medium used is set forth below. The figures indicate grams of the particular component per liter of distilled water. i l I After eight days of stationary incubation at 37 (2., the surface mycelium was removed from each-flask and dis carded and the culture broths were filtered throughamillipore filter (0.45) and pooled. 1 Freeze drying of a sample of the pooled brot hs yields an off-white solid, in an amount of about 13 milligrams per milliliter of liquid. Said solid consists of major; amounts of inorganic salts from the culture medium and minor amounts of FWH-775.

A typical method of carrying out the process of the invention under agitated conditions is set forth hereinbelow:

Twelve 500-ml. flasks were each charged with about 200 ml. of enriched Hanks salts solution, having the composition set forth hereinbefore, inoculated with an aqueous spore suspension of the organism and stoppered with loose cotton plugs.

The flasks were placed on a reciprocal shaker, set to operate at about cycles per minute, and incubated for a period of three days at 37 C. under continuous agitation.

After three days, the mycelium which had formed was removed and discarded and the culture broths were filtered through a millipore filter (0.45) and pooled.

Freeze drying of a sample of the pooled broths, which are substantially lighter than broths obtained by the stationary incubation method, yields a white solid, in an amount of about 13 milligrams per milliliters of liquid. As in the previous example, said solid consists of minor amounts of FWH-77S admixed with major amounts of inorganic salts from the culture medium.

The antiviral activity of the culture broths was tested in various laboratory animals.

PROTECTIVE EFFECT OF FWH-775 ON RABBITS INFECTED WITH VACCINIA VIRUS then infected with vaccinia virus by the scarification of the cornea of the left eye (the right eye served as an internal control) and by intradermal inoculations at four points on the back. Each inoculum consisted of approximately. 10 ID of tissue culture-propagated vaccini'a yirus, a, DNAvirus. Following infection, the administra;

TABLE v Rabbits ++++lllllllll Nora: =Infected with vaclnia virus; caused by vacinia virus.

reaction, I Lesions The above experiment was repeated substantiallyas described. The only difference is that in one test (Table VI), the administration of theactive broth was commenced three'days prior to'infe'ction, and in the other test (Table'VII), the administration of the active broth was commenced simultaneously with the infection. In both tests, the administration of the active broth 'was continued until the twelfth day at a daily dosageof 2.0 ml. per animal. In both tests, the treated animals were completely protected.

TABLE VI Rabbits treated with active Control Day broth rabbits Nora-See footnote bottom of Table V.

TABLE VII Control rabbits: I

Norm-See footnote bottom of Table V.

In order to demonstrate the protective action of FWH- 775 in post-infection administration, five rqups'er rabbits were infected with vaccinia virus as described abovel Each group consisted of three rabbits. Group Areceived the first dose of active broth (2.0 ml. per animal) 24. hours after infection, Group B 16 hours, Group C 6 hours and Group D at the same time as infection. Group E served as the control. r

The administration of the active broth to the test animal was continued until the ninth day at a daily dosage of 2.0 ml. per animal.

As indicated in Table VIII, which follows, FWH-775 protected the infected animals even when"adrninistered 24 hours post-infection.

NoTE.See footnote bottom of Table V.

The protective effect of FWH-775 against influenza virus, a RNA virus, in mice was also demonstrated.

Mice, averaging 15 grams in body weight, were infected with a mouse-adapted PR-8 strain of influenza A virus. A 10 percent suspension of infected mouse lung containing approximately 10 ID was administered'to' each animal.

7 Group A received a daily dosage of 0.5 ml. of active broth intraperitoneally beginning three days before infection, Group B beginning two days before infection, Group C beginning one day before infection and Group D at the same time as infection. Group E served as virus control. The administration of the active broth was continued for. seven days after infection, at which time the animals were observed. The results are set forth in Table IX, in terms ofnumber ofmice dead over number of total mice, used in eachexperiment. A mouse was classified as The above" results illustrate the protective effect of FWH-775-in influenza infection in mice. A comparison of the percentageof healthy mice in each group indicates that administration of the active broth beginning three days preinfection gives the maximumprotective effect, whereas administration beginning at time'of infection protected only 58 percent of the test animals. The percentage of healthy animals in 'the test groups (58-78 percent compares very favorably with the percentage of healthy animals in the control group (12 percent).

It was indeed a surprising discovery to find that the material of the present invention which is obtained from a species of As'prgillus niger is capable of protecting warm blooded animals against 'a'nimal viruses. I "I Aspergillulr nigr is a broad :class of saprophytic fungi 'tholcls which are world wide in; distribution and which'liave been classified without regard 'to morphology or other distinguishing characteristics into the'Aspergillus nig'e r group because of the characteristic pigmentation of the conidial heads. The membersof the class'are notall alike and contrasting differences in structural detail readily' distinguish species within the classification. The classencompasses A. carbonarius (Bain.) Thom'with 3.01 recog In all of the 17 groups classified in Aspergillus niger only 8 groups are known to produce anti-bacterial material and only 1 group Aspergillus fumigatus is known to produce antiviral substances, however, these latter antiviral substances have only been found to be effective against bacterial virus as opposed to the animal virus which is destroyed by the antiviral material of the present invention.

What is claimed is:

1. A method of producing an antiviral fermentation broth having as the antiviral component therein a substance identified as FWH-775 which comprises forming an aqueous fermentation culture medium and cultivating the strain of Aspergillus niger A.T.C.C. 16508 under aerobic conditions therein at a temperature of from about 20 C. to about 40 C. until substantial antiviral activity is produced by said organism in said culture medium.

2. A method substantially as described in claim 1 wherein the culture medium consists of a balanced salt solution enriched with 0.5% lactalbumin hydrolysate and 0.1% yeastolate.

3. A method substantially as described in claim 1 in 10 which said cultivation is carried out at stationary incubation for a period of from 6 to 15 days.

4. A method substantially as described in claim 1 in which said cultivation is carried out under agitated incubation conditions for a period of from 2 to 4 days.

5. An antiviral agent identified as FWH-775, produced by a process which comprises cultivating under aerobic conditions the antiviral producing strain of Aspergillus niger A.T.C.C. 16508 in an aqueous culture medium at a temperature of from about 20 C. to about C. until antiviral activity is produced in said culture medium.

References Cited M. Miric: Plant Disease Reporter, vol. 37, No. 3, pp. 157-158 (1953).

ALVIN E. TANENHOLTZ, Primary Examiner T. G. WISEMAN, Assistant Examiner US. Cl. X.R. -81 

